这项工作是由美国国立卫生研究院资助GM68418的支持。

1.细胞培养和转染<ol><li>放的盖玻璃（22毫米×22毫米）在6孔聚苯乙烯板。可选的外套盖玻璃，聚L-赖氨酸，0.1％w / v的，水（见材料/设备清单），以保证有丝分裂细胞上按照以下步骤玻璃盖: 注意:最佳条件，必须对每个细胞系和应用来确定。 <ol><li>用聚-L-赖氨酸，0.1％重量/体积，在无菌条件下涂层培养表面在水（0.4mL毫升/孔的6孔聚苯乙烯板）。轻轻摇动，以确保即使在培养表面涂层。 </li><li> 5分钟后，吸删除解决方案，并彻底冲洗用无菌组织培养级别的水面。 </li><li>引入细胞和中干前至少2小时。 </li></ol></li><li>种子HeLa细胞17或HeLa细胞的Tet-Off细胞上放置在6孔聚苯乙烯板的盖玻璃（22毫米×22毫米）17,22。检查细胞密度为5.4×10 5每孔。培养细胞在具有10％FBS和1％青霉素 - 链霉素的高葡萄糖DMEM中。 注意:为了达到最佳效果，凭经验确定播种用的细胞密度。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中18小时。 注意:在HeLa细胞的Tet-Off细胞的情况下，外源基因的转录是在不存在诱导剂（ 即 ，四环素/多西环素），因此培养细胞无四环素/多西环素的活性，并与pTRM4表达载体（ 表2瞬时转），其转录由TRE启动子调控（见下文）。 </li><li>播种，转染细胞18小时后，如下所示: <ol><li>通过混合1.5微升的siRNA寡使溶液A（20μM的退火的库存; 表1）和/或2.0微克质粒（ 表2）在50微升减少血清的培养基（参见材料/设备的列表），并在室温孵育5分钟。 注意:在此分析中，CA-UTR的siRNA（5&#39;3和&#39;非编码区的siRNA的混合物; 表1）共转染在协议3和4（见讨论）一起使用。 </li><li>通过混合0.75微升转染试剂I（参见材料/设备的列表）在50μl降低血清培养基，并在室温孵育5分钟使B液。 注:可选的步骤是增加了1.0微升转染试剂II（见材料/设备的列表）。 </li><li>混合溶液A和B一起，并在室温孵育15分钟。 </li><li>用PBS洗涤培养细胞一次，然后加入500μl血清培养基减少到6孔聚苯乙烯板的各孔中。添加溶液A和B（ 即 ，RNA和/或DNA -脂质复合物）的混合物中直接向每个单独的井的。 注意:终浓度为3.3微克/毫升（质粒）; 50纳米（siRNA）的。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中4.5小时。介质更改为高糖DMEM机智H 10％FBS和1％青霉素 - 链霉素。 </li><li>孵育在37℃下将细胞在5％的CO 2的气氛中对转染后48-72小时。 注意:对于最佳的结果，固定之前的潜伏期为细胞生长，必须根据经验来确定，和蛋白质消耗和/或表达，必须通过Western印迹分析来确认（参见协议6）。 </li><li>如果有丝分裂细胞分析很感兴趣，固定前添加紫杉醇（10纳米），以培养细胞24小时，或固定前添加TN16（0.5μM）到培养细胞2.5小时。 </li></ol></li></ol> 2.细胞固定和免疫荧光染色检测内源性着丝粒着丝粒蛋白（多聚甲醛固定） <ol><li>固定剂，缓冲液和试剂的制备。 <ol><li>新鲜制备50毫升的在PBS，pH 7.4的4％低聚甲醛溶液中。 <ol><li>加40毫升1-×PBS中至玻璃烧杯中在搅拌板在通风橱。热而stirring至约60℃。 2克低聚甲醛粉末添加到加热的PBS中。 </li><li>通过在总加入1ml的1N NaOH提高缓慢的pH值，因为粉末不会立即溶解。 注:加入NaOH后的溶液清除。 </li><li>一旦多聚甲醛溶解，冷却，滤出液。 </li><li>调节pH值用1N HCl至pH 7.4，并将该溶液用1×PBS的体积至50ml。 注意:溶液的等分试样可以冷冻或贮存在2-8℃下，只要1周。溶液应冰冷或储存在4℃下直到它被使用。 </li></ol></li><li>制备50ml的缓冲液KB1，它由10毫摩尔Tris盐酸，pH为7.5的; 150毫摩尔NaCl; 0.5％BSA;和0.5％的Triton X-100。 注:BSA应新鲜添加。 注:该缓冲区KB系列是基于以前的报告中描述的缓冲KB 1,2,4。 </li><li>制备50ml的缓冲液KB2，它由10毫摩尔Tris-HCl中的，pH为7.5; 150毫摩尔NaCl;和0.5％的BSA。 注:BSA应新鲜添加。 </li><li>制备1ml含有DAPI（50毫微克/毫升）缓冲KB3的。 </li><li>制备100毫升的安装平台，它由1毫克/毫升对苯二胺的; 10％的PBS，90％甘油。 <ol><li>调整为1×PBS中，以9.8的pH值用1N NaOH，在溶液中溶解对苯二胺，然后添加甘油。 </li><li>商店在-80℃1ml等份。避光。 </li></ol></li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次，用PBS。应用PBS到培养孔的侧面，以避免干扰所述细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在PBS中的4％多聚甲醛将细胞在4℃下30分钟。冲洗细胞两次用缓冲液KB2充分地除去残留的4％多聚甲醛。 </li><li> Permeabi丽泽在缓冲器KB1样品在室温30分钟。用缓冲液KB2冲洗细胞一次，并在室温添加缓冲KB2 5分钟以非特异性结合的附加块的站点。 注:缓冲KB1也显著有助于阻止。 </li><li>从使用镊子6孔聚苯乙烯板卸下的盖玻璃（22毫米×22毫米）。使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形周围形成每个盖玻璃样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。安装了新的6孔聚苯乙烯板玻璃盖。 </li><li>稀释的初级抗体对丝粒或动粒蛋白（第1稀释比:100到1:200;也参见材料/设备的列表）和任一抗CENP-B抗体（第1稀释比:400）或抗着丝点抗体（ACA）（1稀释比例:2,000）作为缓冲KB2着丝粒的位置标记。 注意:为了获得最佳效果，最终concentra在该溶液中的一级抗体的灰必须凭经验确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞用缓冲KB2 3次。 </li><li>用缓冲KB2，稀荧光团缀合的第二抗体（第1稀释比:200:100:1）针对每个第一抗体。 注意:为了获得最佳效果，二次抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>施加足够的体积将稀释的次级抗体（ 约 30微升在盖玻璃的下降）到浸入细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次缓冲KB2期间的30分钟（次6分钟洗涤）。 注意:为了达到最佳效果（ 即对细胞的损失最小），最佳的洗涤条件必须确定empiricaLLY。 </li><li>应用含有DAPI（50毫微克/毫升），以浸没细胞样品缓冲KB3的足够体积。在室温下孵育5分钟的细胞样本。冲洗细胞用缓冲KB2 1-2次。 </li><li>装入包含细胞样品到微滑动玻璃盖。 <ol><li>放置在微滑动的中心安装介质的压降。 </li><li>从细胞样品中移除液体，并使用手或镊子，在微滑动的中心样本位置。避免气泡产生。 </li><li>用纸巾去除多余的封固剂。 </li></ol></li></ol> 3.细胞C端的固定和免疫荧光染色标志标记CENP-A蛋白（丙酮固定） <ol><li>制备<ol><li>准备冰冷的75％丙酮。 </li><li>新鲜制备50ml含0.5％脱脂乳和0.5％BSA的PBS（pH 7.4）中的。 </li><li>新鲜制备50ml含0.1％脱脂乳和0.1％BSA的PBS（pH 7.4）中的。 </li><li>制备1ml的PBS（pH 7.4）中含有DAPI（50-100纳克/毫升）。 </li><li>在2.1.5中描述制备100ml的安装介质。 </li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次，用PBS。应用PBS到培养井的侧面，以避免干扰所述细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在冰冷的75％丙酮的细胞，并孵育细胞在-20℃下10分钟。干细胞在通风橱中在室温下30-60分钟的护罩玻璃。 注意:为了达到最佳效果，需要为固定和细胞干燥的时间长度，必须根据经验来确定。 </li><li>使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形周围形成每个盖玻璃样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。 </li><li>块nonspecIFIC加入含有的PBS 0.5％脱脂乳和0.5％的BSA在室温下5分钟的结合在细胞上的网站。 </li><li>使用含有0.1％脱脂乳和0.1％BSA的PBS稀释的抗Flag抗体（1:1000稀释比），或者抗CENP-B抗体（第1稀释比:200）或ACA（1:2,000稀释比），为着丝粒的位置标记。 注意:为了获得最佳效果，第一抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次用封闭缓冲期间的30分钟。 注:细胞可以用PBS漂洗。对于最佳结果（ 即 ，为了避免细胞的损失），最佳的洗涤条件，必须根据经验来确定。 </li><li>稀释荧光团缀合的第二抗体（第1稀释比:200:100:1）针对在含有0.1％脱脂乳和0.1％BSA的PBS中的每个主抗体。 注意:为了获得最佳效果，二次抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的二级抗体的足量（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞2次，用含有0.1％脱脂乳和0.1％BSA的PBS。 注意:单独的PBS也可用于洗涤细胞。 </li><li>适用的PBS足够体积含有DAPI（50-100纳克/毫升），以浸没细胞样本。在室温下孵育5分钟的细胞样本。冲洗细胞1-2次，用PBS。 </li><li>安装在2.11中所述含细胞样品到微滑动玻璃盖。 </li></ol> 4.细胞N端的固定和免疫荧光染色标志标记CENP-A蛋白（甲醇固定） <ol><li>制备<ol><li>准备冰冷的甲醇。</li><li>制备的TBS含有4％山羊血清（pH 7.4）中。 </li><li>制备1毫升含TBS DAPI（50-100毫微克/毫升）（pH值7.4）。 </li><li>在2.1.5中描述制备100ml的安装介质。 </li></ol></li><li>在48-72小时后转染（见方案1），进行细胞固定除去通过抽吸培养基中。冲洗细胞一次用TBS。适用的TBS到培养孔的一侧，以避免扰乱细胞的表面上。 注:细胞固定的最佳时间点，必须根据经验来确定。 </li><li>固定在冰冷的甲醇中的细胞，并孵育细胞在-20℃下6分钟。冲洗细胞两次，用TBS充分除去残留的甲醇。 </li><li>使用疏水屏障笔（见材料/设备清单），画一个浅绿色的方形或圆形创建一个围绕每个玻璃罩样品疏水性障碍。请勿触摸或太靠近细胞与疏水屏障笔。 </li><li>块上的CEL非特异性结合位点用含4％山羊血清加入TBS LS。在室温下孵育10分钟。 </li><li>稀释的抗Flag抗体（1:1000稀释）和任一抗CENP-B抗体（第1稀释比:200）:在含有4％山羊血清的TBS（2,000稀释比1）作为着丝粒的位置标记或ACA 。 注意:为了获得最佳效果，第一抗体在此溶液中的最终浓度必须根据经验来确定。 </li><li>应用稀释的初级抗体的足够的体积（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞5次用封闭缓冲期间的30分钟。对于最佳结果（ 即 ，细胞的损失最小），最佳的洗涤条件，必须根据经验来确定。 </li><li>稀释荧光团缀合的第二抗体（第1稀释比:200:100:1）在含有4％山羊血清的TBS针对每个初级抗体。 注意:为了达到最佳效果，国际泳联在该溶液中的二级抗体l的浓度必须根据经验来确定。 </li><li>应用稀释的二级抗体的足量（ 约 30微升）沉浸细胞样品。在37℃下孵育细胞样品1小时。冲洗细胞3次，用封闭缓冲液。 </li><li>应用含有DAPI（50-100纳克/毫升）浸入细胞样品的TBS的足够体积。在室温下孵育5分钟的细胞样本。冲洗细胞1-2次用TBS。 </li><li>装入包含在2.11中所述的细胞样品到所述微滑动玻璃盖。 </li></ol> 5.免疫荧光图像观察，采集，定量和分析<ol><li>通过配有一个63X和100X油浸镜头，外部紧凑的光源，和一个数字CCD照相机的机动荧光显微镜观察细胞的样品。 </li><li>进行图像采集和处理，包括反褶积，使用软件A，或软件B1和B2（见材料/设备的列表）。请参阅补充编码文件（5.2.1）中的软件A.对于软件B1和B2使用的所有命令使用的所有命令，请参阅（5.2.2）的补充代码文件。 </li><li>用前述方法23-25 ​​量化着丝粒动粒蛋白的信号（ 例如 ，基本维持在着丝粒CENP-A的信号）具有以下稍作修改: <ol><li>在着丝粒动粒蛋白的选择区域内，以及背景如下: <ol><li>有丝分裂细胞:（着丝粒着丝粒区）选择区域与用DAPI染色体重叠;染色体外，但在相同的单细胞内（ 即细胞质区）（背景区域）和面积。 </li><li>相间细胞:（着丝粒着丝粒区）选择区域与染色用DAPI重叠; （背景区）和区外的染色质，但相同的单细胞内（&lt;EM&gt;即细胞内区域）。 注:另见补充编码文件（5.2.1.4.3）或（5.2.2.6.4）的区域选择与软件A或软件B2分别。 </li></ol></li><li>使用软件A或B.对于此任务，使用下面的公式量化的着丝粒剩余信号的百分比: 在着丝粒动粒剩余着丝粒动粒蛋白的信号 <img alt="公式1" src="/files/ftp_upload/53732/53732equation1.jpg" /> 其中s是所选择的区域，这是由ACA或CENP-B染色证实的信号亮度; b是背景信号的亮度; R 样品是用于siRNA（S）处理的细胞的参照ACA或CENP-B信号;和右 Ctrl是参考ACA或CENP-B信号为吕克的siRNA转染的细胞。 注意:在此分析，以前的报告中描述CENP-B信号被用作参考CUL4A信号和RBX1。 <sup&gt; 17 <ol><li>使用这种计算Excel文件复制后从上述信号定量软件粘贴的原始数据。另见补充编码文件:（5.2.1.4）和（5.2.2.6）了解详细信息。计算的一个例子示于表3中 。 </li></ol></li><li>分析至少20个细胞，以消除在染色和图像采集每个测量电平变化。可以选择使用剩余的着丝粒着丝粒信号的"平均值"为每一个分析单元，以这些价值观不同的着丝粒着丝粒蛋白中进行比较。 </li></ol></li></ol>总蛋白的6印迹分析<ol><li>悬浮细胞在变性缓冲液A（20mM的Tris-盐酸，pH为7.4; 50毫摩尔NaCl; 0.5％的Nonidet P-40的0.5％脱氧胆酸盐; 0.5％SDS; 1mM EDTA中;以及完整无EDTA蛋白酶抑制剂混合物）， 26受该悬浮液以声波处理和冻融过程，并测量蛋白质浓度如下: <ol><li>对于超声处理过程中，加50微升缓冲液A从在48-72小时后转染6孔聚苯乙烯板的两井采集的细胞（参见方案1）。操作装有干扰物喇叭和微尖端超声波仪（参见材料/设备的列表），每一个样品合计15秒的间歇脉冲持续时间（占空比50％）。 </li><li>对于冻融过程中，冷冻用液氮细胞，并在室温解冻细胞。 </li><li>使用商业蛋白质检测试剂I或II测量蛋白浓度（见材料/设备的列表）。 注意:裂解物在与任一试剂I或II的蛋白质浓度的测定1:10的比例稀释。在该稀释中，存在于缓冲液A中的SDS示出了在该测定很少或没有干扰。 </li></ol></li><li>混合含有20-30微克总蛋白的裂解物与2×或4×SDS-PAGE上样缓冲液。27煮沸的样品5分钟，然后加载它们在12.0％-15.0％变性电泳SDS-聚丙烯酰胺凝胶。 </li><li>通过使用先前描述的Western印迹方法转移通过SDS-PAGE分离到PVDF膜上的蛋白质。17,24,27-31 <ol><li>方框用5％无脂奶在膜上的非特异性结合位点在1×PBS中，然后孵育稀释的初级抗体的溶液的膜在RT 1小时。请参阅各主要抗体的详细信息（ 例如，稀释比例）材料/设备清单。 </li><li>用PBS-T缓冲液洗涤该膜3-4次（每3到5分钟孵育振荡）后（1×PBS和0.1％吐温-20），孵育在近红外（IR）的混合物，该膜荧光染料缀合的二级抗体（第1稀释比:20,000），DyLight缀合的第二抗体（1稀释比:20,000），和/或辣根过氧化物酶（HRP）缀合的二级抗体（在PBS-T稀释; diluti上的比例为1:在RT 1小时10,000）。请参阅各二级抗体的详细信息（ 例如，稀释比例）材料/设备清单。 </li></ol></li><li>洗膜3次，然后扫描该膜，以分析与红外成像系统和/或用于免疫印迹检测的化学发光成像的蛋白质（参见材料/设备的列表）。 <ol><li>对于使用红外成像系统，请参阅（6.4.1）的补充代码文件。 </li><li>对于使用化学发光成像仪，见（6.4.2）的补充代码文件。使用超灵敏化学发光增强（ECL）底物（见材料/设备清单）这个系统。 </li></ol></li></ol>

作者宣称，他们没有竞争的经济利益。

近年来，许多研究已经在着丝粒动粒生物学为固定细胞开发了不同的定量显微镜测定。42进展通常需要的蛋白质的着丝粒特异性或特异性着丝点功能的理解，其中亚细胞时空调控反映不断变化的功能在细胞周期的这些蛋白质。因此，这里我们开发IIF染色和定量的IIF分析特异性分析内源性和外源性CENP-A蛋白的相对水平，这可以适用不同处理的样品中的方法。目前，我们实现了内源性CENP-I，CEN...

"着丝粒"被经典定义为遗传学抑制减数分裂重组的区域和以后确认为有丝分裂染色体的主收缩，其中有丝分裂期间起着准确的染色体分离中起重要作用。 "着丝粒"被描述为结合到微管在着丝粒的表面上，所揭示的电子显微镜的多层结构; "着丝粒"后来被定义为在本地化有丝分裂的着丝粒的大分子复合物。尽管脊椎动物中的着丝粒DNA序列的戏剧性发散，动粒的结构和组成是高度保守的。需要有丝分裂过程中染色体分离友们纺锤体微管和动粒之间的动态互动，在着丝粒动粒功能导致非整倍体，从而癌症的缺陷。 在大多数真核生物着丝粒没有定义的DNA序列，而是由包括171 bp的α卫星DNA分子的重复DNA白斑样的大阵列（0.3-5兆）的。除了 ​​在芽殖酵母，着丝粒的身份不是由DNA序列，但由包含组蛋白H3变体CENH3特殊核小体存在下实现（着丝粒蛋白A [CENP-A〕在人类中）。5 CENP-A的核小体定位于哺乳动物着丝粒7并绑定到171 bp的α卫星DNA分子的内板。主动要求着丝粒CENP-含A-核直接组成着丝粒​​相关网络（CCAN）和动粒蛋白的募集，它们共同规范染色体通过纺锤体检查点的有丝分裂纺锤体和随后的周期进程的附件。 另外，在上述证据的光，CENP-A已被提出是着丝粒8的后生标记;然而，这个过程由CENP-A结合我n要着丝粒DNA并负责此掺入的因素尚未充分表征。短丝粒靶向域（CATD）驻留在组蛋白折叠CENP-A的区域和替换H3与CATD相应区域的是足以直接H3到着丝粒。9几项研究建议的翻译后的功能角色修改CENP-A 12-16（PTM）;然而，CENP-A的招募这些翻译后修饰的至着丝粒的分子机制尚未阐明。我们以前报道，CUL4A-RBX1-COPS8 E3连接酶的活性需要CENP-A到着丝粒CENP-A K124泛素化和本地化。17 着丝粒蛋白的发现和鉴定，导致对染色体分离的新见解。18 100多名动粒的成分已通过各种途径在脊椎动物细胞中鉴定。19,20的下在荧光显微镜的着丝点是如何组装和功能也来自各着丝粒动粒蛋白和蛋白质网络的细胞功能的细胞内的表征地位。19直接可视化和先进的成像方法提供着丝粒着丝粒组件非凡分辨率及允许直接观察着丝粒和着丝粒的特定的分子组件。此外，使用特异性抗体间接免疫荧光的方法（IIF）染色对这些观察结果是至关重要的。然而，尽管关于IIF协议，很少在着丝粒特异性着丝粒蛋白质的细节问题的讨论的许多报告。1-4。因此，显影和报告IIF染色和定量IIF测定方法具体分析每个着丝粒动粒蛋白是极其重要的。在IIF染色，应该与染色方案进行，以避免所感兴趣的蛋白的损失或单元的其余部分。然而，定影破坏抗原位点偶尔，以及不同的抗体-抗原组合与一种固定剂不佳工作，但很好地与另一种，21和固定剂的选择在很大程度上取决于所感兴趣的蛋白（多个）。因此，不同的固定剂的方法是在着丝粒动粒蛋白IIF染色至关重要。 间接免疫荧光（IIF）染色和测定以解​​决内源性着丝粒动粒蛋白质，包括CENP-A和Flag标记的外源性​​CENP-A蛋白，以及在人类细胞中这些蛋白质的定量本地化这里优化方法已被开发出来。这些方法可以应用到在其它物种的着丝粒，动粒蛋白质的分析。

immunofluorescence analysis of endogenous and exogenous centromere-kinetochore proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

内源性CENP-A的免疫荧光分析支持CUL4A-E3连接酶需要CENP-A的本地化着丝粒的假说 我们最近的研究表明，CUL4A-RBX1-COPS8 E3连接酶的活性需要在CENP-A和CENP-A至着丝粒的本地化赖氨酸124（K124）的泛素化17最初，相间着丝复合体（ICEN）被隔离抗CENP-A:原生染色质免疫沉淀，32-34，它一直推测，一些ICEN蛋白质可能在定位CENP-A至着丝粒的作用。因此，进行...

Watch this Scientific Journal Video about Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins at JoVE.com

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

内源性的免疫荧光分析及外源着丝粒着丝粒蛋白

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

immunofluorescence-analysis-endogenous-exogenous-centromere

Research

JoVE Journal

Biology

15.1K 次观看.  Nationwide Children's Hospital. Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

视频: Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

科研

生物学

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells. While fluorescent 
proteins such as green fluorescence protein (GFP) have been extensively used as fusion partners to proteins to track the properties of a protein of interest1, recent 
developments with smaller tags enable new functionalities of proteins to be examined in cells such as conformational change and protein-association 2, 3. One small 
tag system involves a tetracysteine motif (CCXXCC) genetically inserted into a target protein, which binds to biarsenical dyes, ReAsH (red fluorescent) and FlAsH 
(green fluorescent), with high specificity even in live cells 2. The TC/biarsenical dye system offers far less steric constraints to the host protein than 
fluorescent proteins which has enabled several new approaches to measure conformational change and protein-protein interactions 4-7. We recently developed 
a novel application of TC tags as sensors of oligomerization in cells expressing mutant huntingtin, which when mutated aggregates in neurons in Huntington 
disease 7. Huntingtin was tagged with two fluorescent dyes, one a fluorescent protein to track protein location, and the second a TC tag which only 
binds biarsenical dyes in monomers. Hence, changes in colocalization between protein and biarsenical dye reactivity enabled submicroscopic oligomer 
content to be spatially mapped within cells. Here, we describe how to label TC-tagged proteins fused to a fluorescent protein (Cherry, GFP or CFP) 
with FlAsH or ReAsH in live mammalian cells and how to quantify the two color fluorescence (Cherry/FlAsH, CFP/FlAsH or GFP/ReAsH combinations).

The biarsenical dyes FlAsH and ReAsH bind specifically to tetracysteine motifs in proteins and can selectively label proteins in live cells. Recently this labeling strategy has been used to develop sensors for different protein conformations or oligomeric states. We describe the labeling approach and methods to quantitatively analyze binding.

ReAsH / FLASH标签和图像分析Tetracysteine​​传感器蛋白在细胞中

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

钙调蛋白结合蛋白的下拉

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

从富集组织及消化细胞外基质蛋白的成肽质谱分析

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain slices and neuronal cell cultures. Recently, this technology has been applied to the whole organs. Advances in sensor design have made &#160;possible the measuring of&#160;cell signaling in blood-perfused in vivo kidneys. The present protocols list the steps needed to measure ATP and H2O2 signaling in the rat kidney interstitium. Two separate sensor designs are used for the ex vivo and in vivo protocols. Both types of sensor&#160;are coated with&#160;a thin enzymatic biolayer on top of a permselectivity layer to give fast responding, sensitive and selective biosensors. The permselectivity layer protects the signal from the interferents in biological tissue, and the enzymatic layer utilizes&#160;the sequential catalytic reaction of glycerol kinase and glycerol-3-phosphate oxidase in the presence of ATP to produce H2O2. The set of sensors used for the ex vivo studies further detected analyte by oxidation of H2O2 on a platinum/iridium (Pt-Ir) wire electrode. The sensors for the&#160;in vivo studies are instead based on the reduction of H2O2 on a mediator coated gold electrode designed for blood-perfused tissue. Final concentration changes&#160;are detected by real-time amperometry followed by calibration to known concentrations of analyte. Additionally, the specificity of the amperometric signal can be confirmed by the addition of enzymes such as catalase and apyrase that break down H2O2 and ATP correspondingly. These sensors also rely heavily on accurate calibrations before and after each experiment. The following two protocols establish the study of real-time detection of ATP and H2O2 in kidney tissues, and can be further modified to extend the described method for use in other biological preparations or whole organs.

Use of Enzymatic Biosensors to Quantify Endogenous ATP or H2O2 in the Kidney

Enzymatic microelectrode biosensors enable real-time measurements of extracellular cell signaling in biologically-relevant concentrations. The following protocols extend the applications of biosensors to the ex vivo and in vivo detection of ATP and H2O2 in the kidney.

利用酶生物传感器的量化内源性ATP或H<sub&gt; 2</sub&gt; 0<sub&gt; 2</sub&gt;在肾脏

Enzymatic microelectrode biosensors have been widely used to measure extracellular signaling in real-time. Most of their use has been limited to brain ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

从冷冻芯的外层部分外源物质的去除探讨古代生物群落里面窝藏

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated &#945; subunit (HIF-1&#945;) and constitutively expressed &#946; subunit (HIF-1&#946;) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1&#945; stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1&#945; and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1&#945; and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

Here we describe a co-immunoprecipitation protocol to study protein-protein interactions between endogenous nuclear proteins under hypoxic conditions. This method is suitable for demonstration of the interactions between transcription factors and transcriptional co-regulators at hypoxia.

缺氧条件下培养细胞内源性核蛋白的共免疫沉淀测定

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

可视化和跟踪活果蝇黑色素细胞卵室的内源性 mRNA

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...